基于腺相关病毒传染的生产过程中的异质性限制了生产效率的提高
Brian Ladd1,2, Sofia Tunmats1,2, Torbjörn Gräslund1,3
1AdBIOPRO Competence Centre for Advanced Bioproduction, Stockholm, Sweden.
Scientific reports
|November 4, 2025
概括
重组腺相关病毒 (rAAV) 生产的过渡性转染是低效的,只有很小一部分细胞表达必要的基因. 优化细胞循环调节和免疫反应可以显著提高基因治疗的rAAV产量.
科学领域:
- 生物技术是生物技术.
- 基因治疗 基因治疗
- 分子生物学分子生物学
背景情况:
- 重组腺相关病毒 (rAAV) 载体对于基因疗法至关重要,但目前的生产方法面临挑战.
- 过渡性转染是一种常见的方法,其产量低且不一致,增加了基因疗法治疗的成本.
研究的目的:
- 调查基于短暂转染的rAAV生产中的低产量.
- 通过使用先进的表征方法,识别RAAV生产过程中的瓶.
主要方法:
- 使用聚乙烯胺 (PEI) 进行HEK293T细胞的三重传染,用于rAAV9生产.
- 在转染后的不同时间点对大量和单细胞转录组学的分析.
- 流细胞计和定量PCR (qPCR) 用于囊和标位分析.
主要成果:
- 很大一部分细胞 (46%) 无法表达 rAAV 生产所需的三个质粒中的基因.
- 在表达所有等离子体基因的细胞中,只有8%的细胞表达高,只有3%的细胞含有组装的rAAV9囊体.
- 单细胞转录组学揭示了细胞周期阶段 (S-阶段) 和与G2-M过渡,免疫反应和高表达细胞中蛋白质展开相关的差异性基因表达.
结论:
- 为了产生rAAV,暂时的转染具有显著的瓶,只有不到5%的细胞有效地产生rAAV.
- 提高rAAV生产标位需要增加高生产细胞的比例.
- 针对细胞循环调节,免疫反应抑制和蛋白质错折的策略可能会提高rAAV产量和基因疗法的可访问性.
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